Aberrant angiogenesis plays a role in the pathogenesis of numerous diseases, including malignant, ischemic, inflammatory and immune disorders (reviewed in Matter, Drug Discovery Today, 6:1005-1024 (2001); Yancopoulos et al, Nature, 407:242-248 (2000); Carmeliet, Nat. Med., 9(6):653-660 (2003); Ferrara, Semin. Oncol., 29(6 Suppl 16):10-14 (2002)). Vascular Endothelial Growth Factor (VEGF), an angiogenesis regulator, plays a central role in angiogenesis. In particular, VEGF is an important factor in the pathogenesis of cancers, diabetic retinopathy (DR), and exudative macular degeneration (reviewed in Tandle et al., Clin. Adv. in Hemat. and Oncol., 1(1):41-48 (2003); Ferrara et al., Nat. Med., 5(12):1359-1364 (1999); Matter, supra; Carmeliet supra; Kerbel et al., Nat. Rev. Cancer, 2(10):727-739 (2002); Witmer et al., Prog. Retin. Eye Res., 22(1):1-29 (2003); Clark et al., Nat. Rev. Drug Discovery, 2:448-459 (2003); Ferrara (2002), supra; Thomas, J. Biol. Chem., 271:603-606 (1996); Gerber et al., Development, 126:1149-1159 (1999); which are hereby incorporated by reference), the last two of which are leading causes of blindness in the United States.
The expression of VEGF is regulated by a number of factors and agents including cytokines, growth factors, steroid hormones and chemicals, and mutations that modulate the activity of oncogenes such as ras or the tumor suppressor gene VHL (Maxwell et al., Nature, 399:271-275 (1999); Rak et al., Cancer Res., 60:490-498 (2000)). In part, VEGF expression is regulated after transcription by sequences in both the 5′- and 3′-untranslated regions (UTRs) of its mRNA (Ikeda et al., J. Biol. Chem., 270:19761-19766 (1995); Stein et al., Mol. Cell. Biol., 18:3112-3119 (1998); Levy et al. J. Biol. Chem., 271:2746-2753 (1996); Huez et al., Mol. Cell. Biol., 18:6178-6190 (1998); Akiri et al., Oncogene, 17:227-236 (1998)). The VEGF 5′ UTR is unusually long and GC rich, and it contains an internal ribosomal entry site (IRES) that is reported to mediate a unique, cap-independent mode of translation initiation. The VEGF 3′ UTR harbors multiple AU-rich stability determinants that have been shown to be associated with VEGF mRNA turnover rates.
Initiation of translation of the VEGF mRNA is reported to be unique under hypoxic conditions in that it is mediated via an internal ribosome entry site (IRES) within the VEGF 5′ UTR (Stein et al., supra; Levy et al., supra; Huez et al., supra; Akiri et al., supra). Under hypoxic conditions, cap-dependent translation is dramatically impaired and the translation of the VEGF mRNA occurs through its cap-independent IRES. Initiation of translation of most eukaryotic mRNA is cap-dependent. IRES-mediated translation initiation becomes predominant when components of the translation initiation complex become rate-limiting, e.g., during hypoxia (Mitchell et al., Mol. Cell., 1(3):757-771 (2003)).
Several investigators have used in vitro and bicistronic strategies, often in conjunction with deletion mutants, to study regulation of VEGF. Prats and colleagues reported the occurrence of cap-independent translation of human VEGF by virtue of an IRES. From these studies, they postulated the presence of two IRESs, a first IRES (IRES A) located within 300 nucleotides of the initiation codon and a second IRES (IRES B) located in the upstream half of the 5′-UTR. Huez et al., supra. In Stein et al., supra., deletion mutants in dicistronic and monocistronic constructs were used to identify sequences of the VEGF 5′ UTR required for maximal IRES activity. Keshet and colleagues reported an increase in IRES activity from a 163-nucleotide sequence derived from a VEGF 5′ UTR, which is possibly an artifact from RT-PCR amplification, relative to the entire full-length VEGF 5′ UTR. Stein et al., supra. Goodall and colleagues reported a deletion analysis of IRES residues toward the 3′ end of the mouse VEGF 5′ UTR, and speculated that, for optimal IRES activity, the upstream half of the VEGF 5′ UTR is necessary. Miller et al., supra.
The present invention provides, for the first time, a negative regulator of post-transcriptional regulation (NeRP) located in the 5′ UTR of VEGF. Removal of a NeRP from the VEGF 5′ UTR results in increased translation of an operably linked gene dependent on the presence of a PTCRE of the present invention.